[Direct expression of the pig testicular 3 alpha/beta(20 beta)- hydroxysteroid dehydrogenase cDNA using baculovirus expression system].
Yakugaku Zasshi
; 115(4): 344-50, 1995 Apr.
Article
en Ja
| MEDLINE
| ID: mdl-7602499
ABSTRACT
The direct expression of the pig testicular 3 alpha/beta(20 beta)- hydroxysteroid dehydrogenase (HSD) cDNA using a baculovirus expression system was investigated. A minimum essential cDNA containing the coding region of 3 alpha/beta(20 beta)-HSD was amplified by polymerase chain reaction (PCR) to extend the DNA linker including the Bam HI restriction site in the both ends of the cDNA. As the template, 3 alpha/beta(20 beta)-HSD cDNA, pBS 52, which was subcloned to Bluescript II was used. The PCR fragment was ligated to Bam HI-cut transfer plasmid (pBlueBac III). Recombinant transfer plasmid (pBlueBac-20 beta) constructed was cotransfected into Spodoptera frugiperda Sf-9 cells with the baculovirus. After transfection, the recombinant virus was detected by the plaque assay with color selection. The expression of 3 alpha/beta(20 beta)-HSD cDNA was detected by Western blotting and enzyme assay. The expressed protein showed the same molecular weight and immunochemical cross-reaction to the native enzyme. Furthermore, it had 3-keto reductase activity of 3 alpha/beta(20 beta)-HSD for 5 alpha-dihydrotestosterone and 20-keto reductase activity for 17 alpha-hydroxyprogesterone in the presence of NADPH.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Testículo
/
Baculoviridae
/
ADN Complementario
/
Cortisona Reductasa
/
3-Hidroxiesteroide Deshidrogenasas
Límite:
Animals
Idioma:
Ja
Revista:
Yakugaku Zasshi
Año:
1995
Tipo del documento:
Article
País de afiliación:
Japón